PROJECT SUMMARY/ABSTRACT Activation of the ?tissue-protective? cytokine receptor produces cytoprotective, anti-inflammatory, and anti- apoptotic effects in vitro and in vivo in preclinical studies, and has demonstrated therapeutic utility in early clinical studies of the rare neuromuscular disease, Friedreich?s ataxia. This heteromeric receptor, composed of EPOR (erythropoietin receptor) and CSF2RB (colony-stimulating factor 2 receptor beta subunit, also ?beta common receptor,? and CD131), is expressed widely in non-hematopoietic tissues where it is activated by locally produced EPO, especially near sites of oxidative stress and tissue damage. Selective EPOR/CSF2RB activators include recombinant proteins, e.g., EPO derivatives, and pharmacologically active small molecules which increase EPOR and CSF2RB phosphorylation in cells and increase expression of the mitochondrial protein, frataxin, a biomarker for Friedreich?s ataxia. Development of small molecules compared to recombinant proteins provides distinct opportunities for optimization of pharmaceutical properties including receptor sub-type selectivity, pharmacokinetics, and tolerability/safety and has produced promising probe compounds. However, discovery of small molecule cytokine receptor modulators has been challenging and slow to advance, despite substantial efforts to develop technologies for studying the effects of modulators of protein-protein interactions. In Phase I, we developed an innovative proximity-based method for detecting dimerization of the EPOR/CSF2RB receptor extracellular domains using homogeneous formats which are fully compatible with high-throughput screening (?HTS?) and demonstrated modulation by EPO and small molecules. In Phase II, we will focus on three major Aims: (1) continue developing the homogeneous EPOR/CSF2RB receptor oligomerization assay to enable HTS and screen a collection of chemically diverse compounds with which we?ll identify new small molecule leads and establish a receptor-binding SAR; (2) expand homogeneous assays for three closely related cytokine receptors as counter-screens to EPOR/CSF2RB to establish target selectivity; and (3) demonstrate the activity of selective small molecules in cellular models in vitro for EPOR/CSF2RB receptor activation and frataxin increase. Through these studies, we will develop an unprecedented small molecule SAR for receptor binding and pharmacological activity, supporting identification of new candidates for Friedreich?s ataxia.